Isolated genomic polynucleotide fragments from chromosome 10q25.3 that encode human soluble aminopeptidase P

ABSTRACT

The invention is directed to an isolated genomic polynucleotide fragment that encodes human soluble (cytosolic) aminopeptidase P, vectors and hosts containing the fragment and fragments hybridizing to noncoding regions as well as antisense oligonucleotides to these fragments. The invention is further directed to methods of using these fragments to obtain human soluble aminopeptidase P and to diagnose, treat, prevent and/or ameliorate a pathological disorder.

PRIORITY CLAIM

This application is a continuation application of application Ser. No.10/457,715, filed Jun. 9, 2003, the contents of which are incorporatedherein by reference. Application Ser. No. 10/457,715 claims priority toprovisional application Ser. No. 60/386,941, filed Jun. 7, 2002 under 35U.S.C. 119(e), the contents of which are also incorporated herein byreference.

FIELD OF THE INVENTION

The invention is directed to isolated genomic polynucleotide fragmentsthat encode human soluble (cytosolic) aminopeptidase P, vectors andhosts containing these fragments and fragments hybridizing to noncodingregions as well as antisense oligonucleotides to these fragments. Theinvention is further directed to methods of using these fragments toobtain human soluble aminopeptidase P and to diagnose, treat, preventand/or ameliorate a pathological disorder.

BACKGROUND OF THE INVENTION

Chromosome 10 contains genes encoding, for example, thealpha-2A-adrenergic receptor, the beta-1-adrenergic receptor,glyceryl-3-phosphate acyltransferase and G protein-coupled receptorkinase-5. Recently, it has been shown that the soluble (cytosolic)aminopeptidase P gene (XPNPEP1) is disposed at 10q25.3 (Sprinkle et al.Arch. Biochem. Biophys. 378: 51-6, 2000), a gene discussed in furtherdetail below.

Human Soluble Aminopeptidase P

Human soluble aminopeptidase P, an aminoacylprolyl peptidyl hydrolase,catalyzes the removal of the N-terminal amino acid from peptides inwhich the second residue is proline. It is believed to actphysiologically by degrading peptide hormones such as bradykinin andsubstance P. It may also degrade collagen-related peptides that haveN-terminal sequences of the type Xaa-Pro-Hyp-. A functionally-relatedenzyme is membrane-bound aminopeptidase P, the gene for which (XPNPEP2)is disposed at chromosome Xq25 (Sprinkle et al, Genomics 50: 114-6,1998). The membrane-bound enzyme is disposed, via aglycosylphosphatidylinositol lipid anchor, as an ectoenzyme onendothelia and epithelia. Soluble aminopeptidase P is disposedintracellularly in virtually all cell types, including astrocytes,lymphocytes, platelets and chromaffin cells.

OBJECTS OF THE INVENTION

Although cDNA encoding the above-disclosed protein, solubleaminopeptidase P, has been isolated (e.g. see accession no. AF195530),its exact location on chromosome 10q25.3 and exon/intron/regulatoryorganization have not been determined. Furthermore, genomic DNA encodingthe polypeptide has not been isolated. Noncoding sequences play asignificant role in regulating the expression of polypeptides as well asthe processing of RNA encoding these polypeptides.

There is clearly a need for obtaining genomic polynucleotide sequencesencoding the soluble aminopeptidase P polypeptide. Therefore, it is anobject of the invention to isolate such genomic polynucleotidesequences.

There is also a need to develop means for identifying mutations,duplications, translocations, polysomies and mosaicism as may affect thesoluble aminopeptidase P gene.

SUMMARY OF THE INVENTION

The invention is directed to an isolated genomic polynucleotide, saidpolynucleotide obtainable from human chromosome 10 having a nucleotidesequence at least 95% identical to a sequence selected from the groupconsisting of:

-   -   (a) a polynucleotide encoding human soluble aminopeptidase P        depicted in SEQ ID NO:1;    -   (b) a polynucleotide consisting of SEQ ID NO:2, which encodes        human soluble aminopeptidase P depicted in SEQ ID NO:1;    -   (c) a polynucleotide which is a variant of SEQ ID NO:2;    -   (d) a polynucleotide which is an allelic variant of SEQ ID NO:2;    -   (e) a polynucleotide which encodes a variant of SEQ ID NO:1;    -   (f) a polynucleotide which hybridizes to any one of the        polynucleotides specified in (a)-(e) and        a polynucleotide that is a reverse complement to the        polynucleotides specified in (a) to (f) as well as nucleic acid        constructs, expression vectors and host cells containing these        polynucleotide sequences.

The polynucleotides of the present invention may be used to modulatehuman soluble aminopeptidase P levels in subjects (e.g., human patients)in need thereof and thus for the manufacture of a gene therapy for theprevention, treatment or amelioration of a medical condition by addingan amount of a composition comprising said polynucleotide effective toprevent, treat or ameliorate said medical condition.

The invention is further directed to obtaining human solubleaminopeptidase P or variant thereof by

-   -   (a) culturing host cells comprising these sequences under        conditions that provide for the expression of said polypeptide        and    -   (b) recovering said expressed polypeptide.

The polypeptides obtained may be used to produce antibodies by

-   -   (a) optionally conjugating said polypeptide to a carrier        protein;    -   (b) immunizing a host animal with said polypeptide or        peptide-carrier protein conjugate of step (b) with an adjuvant        and    -   (c) obtaining antibody from said immunized host animal.

The invention is further directed to a nucleic acid molecule includingbut not limited to a polynucleotide fragment, antisense oligonucleotideor antisense mimetic comprising a sequence of nucleotides whichspecifically hybridizes to noncoding regions of said polynucleotidesequences of SEQ ID NO:2 (human soluble aminopeptidase P). Thesesequences may be used to modulate levels of human soluble aminopeptidaseP in a subject in need thereof and specifically for the manufacture of amedicament for prevention, treatment or amelioration of a medicalcondition. As defined herein, a “polynucleotide fragment” may be anucleic acid molecule including DNA, RNA and analogs thereof includingprotein nucleic acids and mixtures thereof and may include a probe andprimer. Such molecules are generally of a length such that they arestatistically unique in the genome of interest. Generally, for a probeor primer to be unique in the human genome, it contains at least 14 to16 contiguous nucleotides of a sequence complementary to or identical toa target sequence of interest. These polynucleotide fragments can be 20,30, 50, 100, 150, 500, 600, 1000, 2000 or more nucleic acids long.Probes and primers may also be referred to as oligonucleotides. Asdefined herein, an “antisense oligonucleotide” is a molecule encoding asequence complementary to at least a portion of an RNA molecule. Thesequence is sufficiently complementary to be able to hybridize with theRNA, preferably under moderate or high stringency conditions to form astable duplex or triplex.

The invention is further directed to kits comprising thesepolynucleotides and kits comprising these sequences. In a specificembodiment, the sequence(s) are attached to a substrate. In a specificembodiment, the support is a microarray. The microarray may contain aplurality of sequences hybridizing to non-coding sequences. As definedherein, a “plurality” of sequences is two or more sequences.Alternatively, the microarray comprises non-coding sequences as well ascoding sequences.

In a specific embodiment, the noncoding regions are transcriptionregulatory regions. The transcription regulatory regions may be used toproduce a heterologous peptide by expressing in a host cell, saidtranscription regulatory region operably linked to a polynucleotideencoding the heterologous polypeptide and recovering the expressedheterologous polypeptide.

The polynucleotides of the present invention may be used to detect apathological condition or susceptibility to a pathological condition ina subject comprising

-   -   (a) isolating genomic DNA from said subject;    -   (b) detecting the presence or absence of a variant in said        genomic DNA using a probe or primer derived from a        polynucleotide hybridizing to non-coding region(s) of said human        aminopeptidase P; and    -   (c) diagnosing a pathological condition or susceptibility to a        pathological condition based on the presence or absence of said        variant.

Probes or primers derived from SEQ ID NO:2 (human soluble aminopeptidaseP) may be used to identify variants including but not limited tomutations, duplications, translocations, polysomies and mosaicism on thehuman soluble aminopeptidase P gene and may be used to identify patientswith or having a propensity for conditions in which bradykinin orsubstance P is produced in excess, e.g., inherited angioedema or acutepancreatitis.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to isolated genomic polynucleotide fragmentsthat encode human soluble aminopeptidase P, which in a specificembodiment is the human soluble aminopeptidase P gene, as well asvectors and hosts containing these fragments and polynucleotidefragments hybridizing to noncoding regions, as well as antisenseoligonucleotides to these fragments.

As defined herein, a “gene” is the segment of DNA involved in producinga polypeptide chain; it includes regions preceding and following thecoding region, as well as intervening sequences (introns) betweenindividual coding segments (exons).

As defined herein, “isolated” refers to material removed from itsoriginal environment and is thus altered “by the hand of man” from itsnatural state. An isolated polynucleotide can be part of a vector, acomposition of matter or can be contained within a cell as long as thecell is not the original environment of the polynucleotide.

The polynucleotides of the present invention may be in the form of RNAor in the form of DNA, which DNA includes genomic DNA and synthetic DNA.The DNA may be double-stranded or single-stranded and if single strandedmay be the coding strand or non-coding strand. The human solubleaminopeptidase P gene is 58735 base pairs in length and contains 19exons (see Table 1 below for location of exons). As will be discussed infurther detail below, the gene is situated in genomic clones AL133416and AL354951.

The polynucleotides of the invention have at least a 95% identity andmay have a 96%, 97%, 98% or 99% identity to the polynucleotides depictedin SEQ ID NO:2 as well as the polynucleotides in reverse senseorientation, or the polynucleotide sequences encoding the human solubleaminopeptidase P polypeptide depicted in SEQ ID NO:1.

A polynucleotide having 95% “identity” to a reference nucleotidesequence of the present invention, is identical to the referencesequence except that the polynucleotide sequence may include, onaverage, up to five point mutations per each 100 nucleotides of thereference nucleotide sequence encoding the polypeptide. In other words,to obtain a polynucleotide having a nucleotide sequence at least 95%identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. The query sequence may be an entire sequence, the ORF (openreading frame), or any fragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to anucleotide sequence of the presence invention can be determinedconventionally using known computer programs. A preferred method fordetermining the best overall match between a query sequence (a sequenceof the present invention) and a subject sequence, also referred to as aglobal sequence alignment, can be determined using the FASTDB computerprogram based on the algorithm of Brutlag et al. (Comp. App. Biosci.(1990) 6:237-245). In a sequence alignment the query and subjectsequences are both DNA sequences. An RNA sequence can be compared byconverting U's to T's. The result of said global sequence alignment isin percent identity. Preferred parameters used in a FASTDB alignment ofDNA sequences to calculate percent identity are:

Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identify,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequenceare calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total numbers of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are made forpurposes of the present invention.

A polypeptide that has an amino acid sequence at least, for example, 95%“identical” to a query amino acid sequence is identical to the querysequence except that the subject polypeptide sequence may include onaverage, up to five amino acid alterations per each 100 amino acids ofthe query amino acid sequence. In other words, to obtain a polypeptidehaving an amino acid sequence at least 95% identical to a query aminoacid sequence, up to 5% of the amino acid residues in the subjectsequence may be inserted, deleted, (indels) or substituted with anotheramino acid. These alterations of the reference sequence may occur at theamino or carboxy terminal positions of the reference amino acid sequenceor anywhere between those terminal positions, interspersed eitherindividually among residues in the referenced sequence or in one or morecontiguous groups within the reference sequence.

A preferred method for determining the best overall match between aquery sequence (a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, can bedetermined using the FASTDB computer program based on the algorithm ofBrutlag et al. (Com. App. Biosci. (1990) 6:237-245). In a sequencealignment, the query and subject sequence are either both nucleotidesequences or both amino acid sequences. The result of said globalsequence alignment is in percent identity. Preferred parameters used ina FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

The invention also encompasses polynucleotides that hybridize to thepolynucleotides depicted in SEQ ID NO: 2. A polynucleotide “hybridizes”to another polynucleotide, when a single-stranded form of thepolynucleotide can anneal to the other polynucleotide under theappropriate conditions of temperature and solution ionic strength (seeSambrook et al., supra). The conditions of temperature and ionicstrength determine the “stringency” of the hybridization. Forpreliminary screening for homologous nucleic acids, low stringencyhybridization conditions, corresponding to a temperature of 42 C, can beused, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide; or 40%formamide, 5×SSC, 0.5% SDS). Moderate stringency hybridizationconditions correspond to a higher temperature of 55 C, e.g., 40%formamide, with 5× or 6×SCC. High stringency hybridization conditionscorrespond to the highest temperature of 65 C, e.g., 50% formamide, 5×or 6×SCC. Hybridization requires that the two nucleic acids containcomplementary sequences, although depending on the stringency of thehybridization, mismatches between bases are possible. The appropriatestringency for hybridizing nucleic acids depends on the length of thenucleic acids and the degree of complementation, variables well known inthe art. The greater the degree of similarity or homology between twonucleotide sequences, the greater the value of T_(m) for hybrids ofnucleic acids having those sequences. The relative stability(corresponding to higher T_(m)) of nucleic acid hybridizations decreasesin the following order: RNA:RNA, DNA:RNA, DNA:DNA.

Polynucleotide and Polypeptide Variants

The invention is directed to both polynucleotide and polypeptidevariants. A “variant” refers to a polynucleotide or polypeptidediffering from the polynucleotide or polypeptide of the presentinvention, but retaining essential properties thereof. Generally,variants are overall closely similar and in many regions, identical tothe polynucleotide or polypeptide of the present invention.

The variants may contain alterations in the coding regions, non-codingregions, or both. Especially preferred are polynucleotide variantscontaining alterations which produce silent substitutions, additions, ordeletions, but do not alter the properties or activities of the encodedpolypeptide. Nucleotide variants produced by silent substitutions due tothe degeneracy of the genetic code are preferred. Moreover, variants inwhich 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or addedin any combination are also preferred.

The invention also encompasses allelic variants of said polynucleotides.An allelic variant denotes any of two or more alternative forms of agene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

The amino acid sequences of the variant polypeptides may differ from theamino acid sequences depicted in SEQ ID NO:1 by an insertion or deletionof one or more amino acid residues and/or the substitution of one ormore amino acid residues by different amino acid residues. Preferably,amino acid changes are of a minor nature, that is conservative aminoacid substitutions that do not significantly affect the folding and/oractivity of the protein; small deletions, typically of one to about 30amino acids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter the specific activityare known in the art and are described, for example, by H. Neurath andR. L. Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, as well as these in reverse.

Noncoding Regions

The invention is further directed to polynucleotide fragments containingor hybridizing to noncoding regions of the human soluble aminopeptidaseP gene. These include but are not limited to an intron, a 5′-non-codingregion, a 3′-non-coding region and splice junctions (see Table 1), aswell as transcription factor binding sites (see Table 2). Thepolynucleotide fragments may be a short polynucleotide fragment which isbetween about 20 nucleotides to about 50 nucleotides in length. Suchshorter fragments may be useful for diagnostic purposes. Such shortpolynucleotide fragments are also preferred with respect topolynucleotides containing or hybridizing to polynucleotides containingsplice junctions. Alternatively larger fragments, e.g., of about 50,150, 500, 600, 2000 or about 5000 nucleotides in length may be used.

TABLE 1 Exon/Intron Regions of the Human Soluble Aminopeptidase P gene(reverse strand coding). Nucleotide no. Exons (stop codon 12803-5)Peptide amino acid no. 19 12806-12931 623-582 18 16233-16331 581-549 1717272-17649 548-523 16 18351-18524 522-465 15 19412-19480 464-442 1420986-21045 441-422 13 23147-23218 421-398 12 25409-25489 397-371 1125622-25678 370-352 10 28129-28179 351-335 9 28783-28872 334-305 830373-30582 304-235 7 32007-32087 234-208 6 34185-34283 207-175 536003-36146 174-127 4 36457-36549 126-96  3 39675-39779 95-61 240965-41030 60-39 1 55647-55760 38-1 

TABLE 2 Transcription Factor Binding Sites of the Human SolubleAminopeptidase P Gene. Sites No. of Sites AP1_C 12 AP4_Q5 15 AP4_Q6 6DELTAEF1_01 9 GATA1_06 4 GATA2_02 4 GATA_C 4 LMO2COM_02 8 LYF1_01 14MYOD_Q6 12 MZF1_01 34 NFAT_Q6 10 NKX25_01 27 S8_01 12 SOX5_01 39 TATA_C7 TCF11_01 37 USF_C 18

In a specific embodiment, such noncoding sequences are expressioncontrol sequences. These include but are not limited to DNA regulatorysequences, such as promoters, enhancers, repressors, terminators, andthe like, that provide for the regulation of expression of a codingsequence in a host cell. In eukaryotic cells, polyadenylation signalsare also control sequences.

In a more specific embodiment of the invention, the expression controlsequences may be operatively linked to a polynucleotide encoding aheterologous polypeptide. Such expression control sequences may be about50-200 nucleotides in length and specifically about 50, 100, 200, 500,600, 1000, 2000 or 5000 nucleotides in length. A transcriptional controlsequence is “operatively linked” to a polynucleotide encoding aheterologous polypeptide sequence when the expression control sequencecontrols and regulates the transcription and translation of thatpolynucleotide sequence. The term “operatively linked” includes havingan appropriate start signal (e.g., ATG) in front of the polynucleotidesequence to be expressed and maintaining the correct reading frame topermit expression of the DNA sequence under the control of theexpression control sequence and production of the desired productencoded by the polynucleotide sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted upstream (5′) of andin reading frame with the gene.

The invention is further directed to antisense oligonucleotides andmimetics to these polynucleotide sequences. Antisense technology can beused to control gene expression through triple-helix formation orantisense DNA or RNA, both of which methods are based on binding of apolynucleotide to DNA or RNA. For example, the 5′ coding portion of thepolynucleotide sequence, which encodes the mature polypeptides of thepresent invention, is used to design an antisense RNA oligonucleotide offrom about 10 to 40 base pairs in length. A DNA oligonucleotide isdesigned to be complementary to a region of the gene involved intranscription or RNA processing (triple helix (see Lee et al., Nucl.Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); andDervan et al., Science, 251: 1360 (1991)), thereby preventingtranscription and the production of said polypeptides.

Expression of Polypeptides

Isolated Polynucleotide Sequences

The human chromosome genomic clones of accession number AL133416 andAL354951 have been discovered to contain the human solubleaminopeptidase P gene by Genscan analysis (Burge et al., 1997, J. Mol.Biol. 268:78-94), BLAST2 and TBLASTN analysis (Altschul et al., 1997,Nucl. Acids Res. 25:3389-3402), in which the sequences of AL133416 andAL354951 were compared to the human soluble aminopeptidase P cDNAsequence, accession number AF195530

The cloning of the nucleic acid sequences of the present invention fromsuch genomic DNA can be effected, e.g., by using the well knownpolymerase chain reaction (PCR) or antibody screening of expressionlibraries to detect cloned DNA fragments with shared structuralfeatures. See, e.g., Innis et al., 1990, PCR: A Guide to Methods andApplication, Academic Press, New York. Other nucleic acid amplificationprocedures such as ligase chain reaction (LCR), ligated activatedtranscription (LAT) and nucleic acid sequence-based amplification(NASBA) or long range PCR may be used. In a specific embodiment, 5′- or3′-non-coding portions of the gene may be identified by methodsincluding but are not limited to, filter probing, clone enrichment usingspecific probes and protocols similar or identical to 5′- and 3′-“RACE”protocols which are well known in the art. For instance, a methodsimilar to 5′-RACE is available for generating the missing 5′-end of adesired full-length transcript. (Fromont-Racine et al., 1993, Nucl.Acids Res. 21:1683-1684).

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired human soluble aminopeptidase P gene maybe accomplished in a number of ways. For example, if an amount of aportion of a human soluble aminopeptidase P gene or its specific RNA, ora fragment thereof, is available and can be purified and labeled, thegenerated DNA fragments may be screened by nucleic acid hybridization tothe labeled probe (Benton and Davis, 1977, Science 196:180; Grunsteinand Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). The presentinvention provides such nucleic acid probes, which can be convenientlyprepared from the specific sequences disclosed herein, e.g., ahybridizable probe having a nucleotide sequence corresponding to atleast a 10, and preferably a 15, nucleotide fragment of the sequencesdepicted in SEQ ID NO:2. Preferably, a fragment is selected that ishighly unique to the polypeptides of the invention. Those DNA fragmentswith substantial homology to the probe will hybridize. As noted above,the greater the degree of homology, the more stringent hybridizationconditions can be used. In one embodiment, low stringency hybridizationconditions are used to identify a homologous human solubleaminopeptidase P polynucleotide. However, in a preferred aspect, and asdemonstrated experimentally herein, a nucleic acid encoding apolypeptide of the invention will hybridize to a nucleic acid derivedfrom the polynucleotide sequence depicted in SEQ ID NO:2 or ahybridizable fragment thereof, under moderately stringent conditions;more preferably, it will hybridize under high stringency conditions.

Alternatively, the presence of the gene may be detected by assays basedon the physical, chemical, or immunological properties of its expressedproduct. For example, cDNA clones, or DNA clones which hybrid-select theproper mRNAs, can be selected which produce a protein that, e.g., hassimilar or identical electrophoretic migration, isoelectric focusingbehavior, proteolytic digestion maps, or antigenic properties as knownfor the human soluble aminopeptidase P polypeptide.

A gene encoding human soluble aminopeptidase P polypeptide can also beidentified by mRNA selection, i.e., by nucleic acid hybridizationfollowed by in vitro translation. In this procedure, fragments are usedto isolate complementary mRNAs by hybridization. Immunoprecipitationanalysis or functional assays of the in vitro translation products ofthe products of the isolated mRNAs identifies the mRNA and, therefore,the complementary DNA fragments, that contain the desired sequences.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide sequence containing the exon/intron segments of thehuman soluble aminopeptidase P gene (nucleotides 58735 of SEQ ID NO:2)operably linked to one or more control sequences which direct theexpression of the coding sequence in a suitable host cell underconditions compatible with the control sequences. Expression will beunderstood to include any step involved in the production of thepolypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

The invention is further directed to a nucleic acid construct comprisingexpression control sequences derived from SEQ ID NO: 2 and aheterologous polynucleotide sequence.

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid which are combined and juxtaposed in a manner which would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term expression cassette when the nucleic acid constructcontains all the control sequences required for expression of a codingsequence of the present invention. The term “coding sequence” is definedherein as a portion of a nucleic acid sequence which directly specifiesthe amino acid sequence of its protein product. The boundaries of thecoding sequence are generally determined by a ribosome binding site(prokaryotes) or by the ATG start codon (eukaryotes) located justupstream of the open reading frame at the 5′-end of the mRNA and atranscription terminator sequence located just downstream of the openreading frame at the 3′-end of the mRNA. A coding sequence can include,but is not limited to, DNA, cDNA, and recombinant nucleic acidsequences.

The isolated polynucleotide of the present invention may be manipulatedin a variety of ways to provide for expression of the polypeptide.Manipulation of the nucleic acid sequence prior to its insertion into avector may be desirable or necessary depending on the expression vector.The techniques for modifying nucleic acid sequences utilizingrecombinant DNA methods are well known in the art.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence which is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptionalcontrol sequences which regulate the expression of the polynucleotide.The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, the Streptomyces coelicolor agarase gene (dagA), the Bacillussubtilis levansucrase gene (sacB), the Bacillus licheniformisalpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenicamylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene(amyQ), the Bacillus licheniformis penicillinase gene (penP), theBacillus subtilis xylA and xylB genes, and the prokaryoticbeta-lactamase gene (VIIIa-Komaroff et al., 1978, Proceedings of theNational Academy of Sciences USA 75: 3727-3731), as well as the tacpromoter (DeBoer et al., 1983, Proceedings of the National Academy ofSciences USA 80: 21-25). Further promoters are described in “Usefulproteins from recombinant bacteria” in Scientific American, 1980, 242:74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes encoding Aspergillusoryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillusniger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulansacetamidase, Fusarium oxysporum trypsin-like protease (WO 96/00787),NA2-tpi (a hybrid of the promoters from the genes encoding Aspergillusniger neutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase), and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the Saccharomycescerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiaegalactokinase gene (GAL1), the Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP),and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Otheruseful promoters for yeast host cells are described by Romanos et al.,1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the3′-terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes encoding Aspergillus oryzae TAKA amylase, Aspergillusniger glucoamylase, Aspergillus nidulans anthranilate synthase,Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesencoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C(CYC1), or Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′-terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used inthe present invention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes encoding Aspergillus oryzae TAKA amylase and Aspergillusnidulans triose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from theSaccharomyces cerevisiae enolase (ENO-1) gene, the Saccharomycescerevisiae 3-phosphoglycerate kinase gene, the Saccharomyces cerevisiaealpha-factor, and the Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequencewhich is operably linked to the 3′-terminus of the nucleic acid sequenceand which, when transcribed, is recognized by the host cell as a signalto add polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes encoding Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, and Aspergillus niger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region, whichcodes for an amino acid sequence linked to the amino terminus of thepolypeptide which can direct the encoded polypeptide into the cell'ssecretory pathway. The 5′-end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′-endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not normallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to obtain enhanced secretion of thepolypeptide. However, any signal peptide coding region which directs theexpressed polypeptide into the secretory pathway of a host cell ofchoice may be used in the present invention.

An effective signal peptide coding region for bacterial host cells isthe signal peptide coding region obtained from the maltogenic amylasegene from Bacillus NCIB 11837, the Bacillus stearothermophilusalpha-amylase gene, the Bacillus licheniformis subtilisin gene, theBacillus licheniformis beta-lactamase gene, the Bacillusstearothermophilus neutral proteases genes (nprT, nprS, nprM), or theBacillus subtilis prsA gene. Further signal peptides are described bySimonen and Palva, 1993, Microbiological Reviews 57: 109-137.

An effective signal peptide coding region for filamentous fungal hostcells is the signal peptide coding region obtained from the Aspergillusoryzae TAKA amylase gene, Aspergillus niger neutral amylase gene,Aspergillus niger glucoamylase gene, Rhizomucor miehei asparticproteinase gene, Humicola lanuginosa cellulase gene, or Humicolalanuginosa lipase gene.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region, which codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from theBacillus subtilis alkaline protease gene (aprE), the Bacillus subtilisneutral protease gene (nprT), the Saccharomyces cerevisiae alpha-factorgene, the Rhizomucor miehei aspartic proteinase gene, or theMyceliophthora thermophile laccase gene (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems would include thelac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1system may be used. In filamentous fungi, the TAKA alpha-amylasepromoter, Aspergillus niger glucoamylase promoter, and the Aspergillusoryzae glucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a nucleic acid sequence of the present invention, a promoter,and transcriptional and translational stop signals. The various nucleicacid and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleic acid sequence encoding the polypeptide at such sites.Alternatively, the polynucleotide of the present invention may beexpressed by inserting the nucleic acid sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleic acid sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis, or markers whichconfer antibiotic resistance such as ampicillin, kanamycin,chloramphenicol or tetracycline resistance. Suitable markers for yeasthost cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Aselectable marker for use in a filamentous fungal host cell may beselected from the group including, but not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5Õ-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), as well as equivalents from other species.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell genomeor autonomous replication of the vector in the cell independent of thegenome of the cell.

For integration into the host cell genome, the vector may rely on thepolynucleotide sequence encoding the polypeptide or any other element ofthe vector for stable integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional polynucleotide sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAM§1permitting replication in Bacillus. Examples of origins of replicationfor use in a yeast host cell are the 2 micron origin of replication,ARS1, ARS4, the combination of ARS1 and CEN3, and the combination ofARS4 and CEN6. The origin of replication may be one having a mutationwhich makes its functioning temperature-sensitive in the host cell (see,e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA75: 1433).

More than one copy of a polynucleotide sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the polynucleotide sequencecan be obtained by integrating at least one additional copy of thesequence into the host cell genome or by including an amplifiableselectable marker gene with the nucleic acid sequence where cellscontaining amplified copies of the selectable marker gene, and therebyadditional copies of the nucleic acid sequence, can be selected for bycultivating the cells in the presence of the appropriate selectableagent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga nucleic acid sequence of the invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising anucleic acid sequence of the present invention is introduced into a hostcell so that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote. Useful unicellularcells are bacterial cells such as gram positive bacteria including, butnot limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus stearothermophilus,Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell,e.g., Streptomyces lividans or Streptomyces murinus, or gram negativebacteria such as E. coli and Pseudomonas sp. In a preferred embodiment,the bacterial host cell is a Bacillus lentus, Bacillus licheniformis,Bacillus stearothermophilus or Bacillus subtilis cell. In anotherpreferred embodiment, the Bacillus cell is an alkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may be a eukaryote, such as a mammalian cell (e.g., humancell), an insect cell, a plant cell or a fungal cell. Mammalian hostcells that could be used include but are not limited to human Hela, 293,H9 and Jurkat cells, mouse NIH3t3 and C127 cells, Cos 1, Cos 7 and CV1,quail QC1-3 cells, mouse L cells and Chinese Hamster ovary (CHO) cells.These cells may be transfected with a vector containing atranscriptional regulatory sequence, a protein coding sequence andtranscriptional termination sequences. Alternatively, the polypeptidecan be expressed in stable cell lines containing the polynucleotideintegrated into a chromosome. The co-transfection with a selectablemarker such as dhfr, gpt, neomycin, hygromycin allows the identificationand isolation of the transfected cells.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (asdefined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary ofThe Fungi, 8th edition, 1995, CAB International, University Press,Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al.,1995, supra, page 171) and all mitosporic fungi (Hawksworth et al.,1995, supra). The fungal host cell may also be a yeast cell. “Yeast” asused herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980). The fungal host cell may also be a filamentous fungalcell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are characterized by a mycelial wallcomposed of chitin, cellulose, glucan, chitosan, mannan, and othercomplex polysaccharides. Vegetative growth is by hyphal elongation andcarbon catabolism is obligately aerobic. In contrast, vegetative growthby yeasts such as Saccharomyces cerevisiae is by budding of aunicellular thallus and carbon catabolism may be fermentative.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; and Hinnen et al., 1978, Proceedings of theNational Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. In a specific embodiment, an enzyme assay may beused to determine the activity of the polypeptide. For example, humansoluble aminopeptidase P can be assayed by its ability to release theN-terminal arginine residue from bradykinin or the synthetic substrateArg-Pro-Pro-benzylamide.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

Antibodies

According to the invention, the human soluble aminopeptidase Ppolypeptide produced according to the method of the present inventionmay be used as an immunogen to generate any of these antibodies. Suchantibodies include but are not limited to polyclonal, monoclonal,chimeric, single chain, Fab fragments, and an Fab expression library.

Various procedures known in the art may be used for the production ofantibodies. For the production of antibody, various host animals can beimmunized by injection with the polypeptide thereof, including but notlimited to rabbits, mice, rats, sheep, goats, etc. In one embodiment,the polypeptide or fragment thereof can optionally be conjugated to animmunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpethemocyanin (KLH). Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward the humansoluble aminopeptidase P polypeptide, any technique that provides forthe production of antibody molecules by continuous cell lines in culturemay be used. These include but are not limited to the hybridomatechnique originally developed by Kohler and Milstein (1975, Nature256:495-497), as well as the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96). In an additional embodiment of the invention,monoclonal antibodies can be produced in germ-free animals utilizingrecent technology (PCT/US90/02545). According to the invention, humanantibodies may be used and can be obtained by using human hybridomas(Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or bytransforming human B cells with EBV virus in vitro (Cole et al., 1985,in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).In fact, according to the invention, techniques developed for theproduction of “chimeric antibodies” (Morrison et al., 1984, J.Bacteriol. 159-870; Neuberger et al., 1984, Nature 312:604-608; Takedaet al., 1985, Nature 314:452-454) by splicing the genes from a mouseantibody molecule specific for the human soluble aminopeptidase Ppolypeptide together with genes from a human antibody molecule ofappropriate biological activity can be used; such antibodies are withinthe scope of this invention.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce polypeptide-specific single chain antibodies. An additionalembodiment of the invention utilizes the techniques described for theconstruction of Fab expression libraries (Huse et al., 1989, Science246:1275-1281) to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity for the human solubleaminopeptidase P polypeptide.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab')₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab'fragments which can be generated by reducing the disulfide bridges ofthe F(ab')₂, fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbent assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of a particular polypeptide, one may assay generatedhybridomas for a product which binds to a particular polypeptidefragment containing such epitope. For selection of an antibody specificto a particular polypeptide from a particular species of animal, one canselect on the basis of positive binding with the polypeptide expressedby or isolated from cells of that species of animal.

Immortal, antibody-producing cell lines can also be created bytechniques other than fusion, such as direct transformation of Blymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980); Hammerlinget al., “Monoclonal Antibodies And T-cell Hybridomas” (1981); Kennett etal., “Monoclonal Antibodies” (1980); see also U.S. Pat. Nos. 4,341,761;4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500;4,491,632; 4,493,890.

Substrate

In a specific embodiment, the polynucleotides of the present invention,particularly, the polynucleotide fragments or antisense nucleic acidshybridizing to non-coding regions of SEQ ID NO:2 may be attached to asubstrate. A substrate may be solid or porous, planar or non-planar,unitary or distributed. The polynucleotide may be attached covalently orapplied to a derivatized surface in a chaotropic agent that facilitatesdenaturation and adherence by presumed noncovalent interactions, or somecombinations thereof.

In a more specific embodiment, the substrate is a microarray.“Microarray” as defined herein is a substrate-bound collection of aplurality nucleic acids, hybridization to each of the plurality of boundnucleic acids being separately detectable. The microarray may comprise aplurality of polynucleotides hybridizing to a non coding region of SEQID NO:2. Alternatively the microarray may comprise a polynucleotide(s)hybridizing to said non-coding region and/or coding regions of SEQ IDNO:2.

Uses of Polynucleotides

Diagnostics

Polynucleotide fragments containing noncoding regions of SEQ ID NO:2 maybe used as probes for detecting variants from genomic nucleotide samplesfrom a patient. The variants may be allelic variants or substitution,insertion or deletion nucleotide variants. Genomic DNA may be isolatedfrom the patient. A mutation(s) may be detected by Southern blotanalysis, specifically by hybridizing restriction digested genomic DNAto various probes between 10-500 nucleotides in length, preferablybetween 20-200 nucleotides in length, more preferably between 20-100nucleotides in length and most preferably between 20-50 nucleotides inlength and subjecting to agarose electrophoresis. Alternatively, thesepolynucleotides may be used as PCR primers between about 10-100nucleotides in length and be used to amplify the genomic DNA isolatedfrom the patients. Methods for performing primer-directed amplification(routine or long range PCR) are well known in the art (see, for example,PCR Basics: From Background to Bench, Springer Verlag (2000); Gelfand etal., (eds.), PCR Strategies, Academic Press (1998). Single baseextension (see, for example, U.S. Pat. No. 6,004,744) may be used todetect SNPs. Additionally, primers may be obtained by routine or longrange PCR that yield products containing contiguous intron(s)/exonsequence(s) and products containing more than one exon with interveningintron. The sequence of the amplified genomic DNA from the patient maybe determined using methods known in the art. Such probes may be between20-5000 nucleotides in length and may preferably be between 20-50nucleotides in length.

Thus the invention is thus directed to kits comprising thesepolynucleotide probes. In a specific embodiment, these probes arelabeled with a detectable substance.

In one embodiment, the probes are in solution. In another embodiment,the probes are attached to a substrate. In a specific embodiment, theprobes are contained within a microarray and are separately detectable.

Antisense Oligonucleotides and Mimetics

The antisense oligonucleotides or mimetics of the present invention maybe used to decrease levels of a polypeptide. For example, human solubleaminopeptidase P has been found to be associated with the inactivationof bradykinin, an antimitogenic agent. Therefore, the human solubleaminopeptidase P antisense oligonucleotides of the present inventioncould be used to inhibit cell growth and in particular, to treatvascular stenosis or restenosis.

The antisense oligonucleotides of the present invention may beformulated into pharmaceutical compositions. These compositions may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention, the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀ as found to be effective in invitro and in vivo animal models.

In general, dosage is from 0.01 ug to 10 g per kg of body weight, andmay be given once or more daily, weekly, monthly or yearly, or even onceevery 2 to 20 years. Persons of ordinary skill in the art can easilyestimate repetition rates for dosing based on measured residence timesand concentrations of the drug in bodily fluids or tissues. Followingsuccessful treatment, it may be desirable to have the patient undergomaintenance therapy to prevent the recurrence of the disease state,wherein the oligonucleotide is administered in maintenance doses,ranging from 0.01 ug to 10 g per kg of body weight, once or more daily,to once every 20 years.

Gene Therapy

As noted above, human soluble aminopeptidase P inactivates bradykininand substance P. Therefore, the human soluble aminopeptidase P gene maybe used to modulate or prevent conditions in which bradykinin orsubstance P is produced in excess; notably in acquired or inheritedangioedema or acute pancreatitis.

As described herein, the polynucleotide of the present invention may beintroduced into a patient's cells for therapeutic uses. As will bediscussed in further detail below, cells can be transfected using anyappropriate means, including viral vectors, as shown by the example,chemical transfectants, or physico-mechanical methods such aselectroporation and direct diffusion of DNA. See, for example, Wolff,Jon A, et al., “Direct gene transfer into mouse muscle in vivo,”Science, 247, 1465-1468, 1990; and Wolff, Jon A, “Human dystrophinexpression in mdx mice after intramuscular injection of DNA constructs,”Nature, 352, 815-818, 1991. As used herein, vectors are agents thattransport the gene into the cell without degradation and include apromoter yielding expression of the gene in the cells into which it isdelivered. As will be discussed in further detail below, promoters canbe general promoters, yielding expression in a variety of mammaliancells, or cell specific, or even nuclear versus cytoplasmic specific.These are known to those skilled in the art and can be constructed usingstandard molecular biology protocols. Vectors have been divided into twoclasses:

a) Biological agents derived from viral, bacterial or other sources.

b) Chemical physical methods that increase the potential for geneuptake, directly introduce the gene into the nucleus or target the geneto a cell receptor.

Biological Vectors

Viral vectors have higher transaction (ability to introduce genes)abilities than do most chemical or physical methods to introduce genesinto cells. Vectors that may be used in the present invention includeviruses, such as adenoviruses, adeno associated virus (AAV), vaccinia,herpesviruses, baculoviruses and retroviruses, bacteriophages, cosmids,plasmids, fungal vectors and other recombination vehicles typically usedin the art which have been described for expression in a variety ofeukaryotic and prokaryotic hosts, and may be used for gene therapy aswell as for simple protein expression. Polynucleotides are inserted intovector genomes using methods well known in the art.

Retroviral vectors are the vectors most commonly used in clinicaltrials, since they carry a larger genetic payload than other viralvectors. However, they are not useful in non-proliferating cells.Adenovirus vectors are relatively stable and easy to work with, havehigh titers, and can be delivered in aerosol formulation. Pox viralvectors are large and have several sites for inserting genes, they arethermostable and can be stored at room temperature.

Examples of promoters are SP6, T4, T7, SV40 early promoter,cytomegalovirus (CMV) promoter, mouse mammary tumor virus (MMTV)steroid-inducible promoter, Moloney murine leukemia virus (MMLV)promoter, phosphoglycerate kinase (PGK) promoter, and the like.Alternatively, the promoter may be an endogenous adenovirus promoter,for example the E1 a promoter or the Ad2 major late promoter (MLP).Similarly, those of ordinary skill in the art can construct adenoviralvectors utilizing endogenous or heterologous poly A addition signals.Plasmids are not integrated into the genome and the vast majority ofthem are present only from a few weeks to several months, so they aretypically very safe. However, they have lower expression levels thanretroviruses and since cells have the ability to identify and eventuallyshut down foreign gene expression, the continuous release of DNA fromthe polymer to the target cells substantially increases the duration offunctional expression while maintaining the benefit of the safetyassociated with non-viral transfections.

Chemical/Physical Vectors

Other methods to directly introduce genes into cells or exploitreceptors on the surface of cells include the use of liposomes andlipids, ligands for specific cell surface receptors, cell receptors, andcalcium phosphate and other chemical mediators, microinjections directlyto single cells, electroporation and homologous recombination. Liposomesare commercially available from Gibco BRL, for example, as LIPOFECTIN″and LIPOFECTACE″, which are formed of cationic lipids such as N-[1-(2,3dioleyloxy)-propyl]-n,n,n-trimethylammonium chloride (DOTMA) anddimethyl dioctadecylammonium bromide (DDAB). Numerous methods are alsopublished for making liposomes, known to those skilled in the art.

For example, Nucleic acid-Lipid Complexes—Lipid carriers can beassociated with naked nucleic acids (e.g., plasmid DNA) to facilitatepassage through cellular membranes. Cationic, anionic, or neutral lipidscan be used for this purpose. However, cationic lipids are preferredbecause they have been shown to associate better with DNA which,generally, has a negative charge. Cationic lipids have also been shownto mediate intracellular delivery of plasmid DNA (Felgner and Ringold,Nature 337:387 (1989)). Intravenous injection of cationic lipid-plasmidcomplexes into mice has been shown to result in expression of the DNA inlung (Brigham et al., Am. J. Med. Sci. 298:278 (1989)). See also, Osakaet al., J. Pharm. Sci. 85(6):612-618 (1996); San et al., Human GeneTherapy 4:781-788 (1993); Senior et al., Biochemica et Biophysica Acta1070:173-179 (1991); Kabanov and Kabanov, Bioconjugate Chem. 6:7-20(1995); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Behr, J-P.,Bioconjugate Chem 5:382-389 (1994); Behr et al., Proc. Natl. Acad. Sci.,USA 86:6982-6986 (1989); and Wyman et al., Biochem. 36:3008-3017 (1997).

Cationic lipids are known to those of ordinary skill in the art.Representative cationic lipids include those disclosed, for example, inU.S. Pat. No. 5,283,185; and e.g., U.S. Pat. No. 5,767,099. In apreferred embodiment, the cationic lipid is N₄-spermine cholesterylcarbamate (GL-67) disclosed in U.S. Pat. No. 5,767,099. Additionalpreferred lipids include N4_spermidine cholestryl carbamate (GL-53) and1-(N-4-spermind)-2,3-dilaurylglycerol carbamate (GL-89).

The vectors of the invention may be targeted to specific cells bylinking a targeting molecule to the vector. A targeting molecule is anyagent that is specific for a cell or tissue type of interest, includingfor example, a ligand, antibody, sugar, receptor, or other bindingmolecule.

Invention vectors may be delivered to the target cells in a suitablecomposition, either alone, or complexed, as provided above, comprisingthe vector and a suitably acceptable carrier. The vector may bedelivered to target cells by methods known in the art, for example,intravenous, intramuscular, intranasal, subcutaneous, intubation,lavage, and the like. The vectors may be delivered via in vivo or exvivo applications. In vivo applications involve the directadministration of an adenoviral vector of the invention formulated intoa composition to the cells of an individual. Ex vivo applicationsinvolve the transfer of the adenoviral vector directly to harvestedautologous cells which are maintained in vitro, followed byreadministration of the transduced cells to a recipient.

In a specific embodiment, the vector is transfected intoantigen-presenting cells. Suitable sources of antigen-presenting cells(APCs) include, but are not limited to, whole cells such as dendriticcells or macrophages; purified MHC class I molecule complexed tobeta2-microglobulin and foster antigen-presenting cells. In a specificembodiment, the vectors of the present invention may be introduced intoT cells or B cells using methods known in the art (see, for example,Tsokos and Nepom, 2000, J. Clin. Invest. 106:181-183).

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Various references are cited herein, the disclosure of which areincorporated by reference in their entireties.

1. An isolated genomic polynucleotide, wherein said polynucleotide isisolated from human chromosome 10 and comprises a polynucleotideidentical to the sequence of nucleotides from position 12803 to position55760 of SEQ ID NO:2, and a polynucleotide which is the complement ofsaid polynucleotide and that encodes a polypeptide having aminoacylprolyl peptidyl hydrolase activity.
 2. A nucleic acid constructcomprising the isolated polynucleotide of claim
 1. 3. An expressionvector comprising the nucleic acid construct of claim
 2. 4. An isolatedrecombinant host cell comprising the nucleic acid construct of claim 2.5. A method for obtaining a polypeptide having aminoacyl prolyl peptidylhydrolase activity comprising (a) culturing the recombinant host cell ofclaim 4 under conditions that provide for the expression of saidpolypeptide, and (b) recovering said expressed polypeptide.
 6. Acomposition comprising the polynucleotide of claim 1 and a carrier.